Mouse Epidermal Growth Factor (mEGF) stimulates cellular proliferation and differentiation in infant mice and in a wide variety of cell cultures. Establishing the sites of synthesis and the conditions effecting the secretion of this polypeptide will require assays with extreme sensitivity. The recent development in this laboratory of the ultrasensitive enzymatic radioimmunoassay (USERIA), significantly more sensitive than RIA, provides the potential of the necessary sensitivity. A modification of the enzyme-linked immunoabsorbent assay (ELISA) has been employed to determine the preliminary conditions for applying the USERIA to the study of mEGF. Mouse EGF is coated onto the wells of microtiter plates and reacted with specific antibody. Heterologous antibody conjugated to alkaline phosphatase is then added. Subsequently, a colorimetric substrate (ELISA) or tritiated adenosine-5'-monophosphate (USERIA) is added as substrate to quantitate the results. A variety of experimental designs have been used to determine the major requirements for optimum conditions in a competitive USERIA.